CTLA-4 is a member of the immunoglobulin superfamily, which is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA-4 is similar to the T-cell costimulatory protein CD28, and both molecules bind to CD80 and CD86 on antigen-presenting cells. CTLA4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA-4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules (i.e. CD80 and CD86). Multiphoton microscopy studies observing T-cell motility in intact lymph nodes gave evidence for the so called ‘reverse-stop signaling model’. In this model CTLA 4 reverse the classic TCR-induced ‘stop signal’ needed for firm contact between T cells and antigen-presenting cells (APCs).
The CTLA-4 protein contains an extracellular V domain, a transmembrane domain, and a cytoplasmic tail. Alternate splice variants, encoding different isoforms, have been characterized. The membrane-bound isoform functions as a homodimer interconnected by a disulfide bond, while the soluble isoform functions as a monomer. The intracellular domain is similar to that of CD28, in that it has no intrinsic catalytic activity and contains one YVKM motif able to bind PI3K, PP2A and SHP-2 and one proline-rich motif able to bind SH3 containing proteins. The first role of CTLA-4 in inhibiting T cell responses seem to be directly via SHP-2 and PP2A dephosphorylation of TCR-proximal signalling proteins such as CD3 and LAT. CTLA-4 can also affect signalling indirectly via competing with CD28 for CD80/86 binding. CTLA-4 can also bind PI3K, although the importance and results of this interaction are uncertain.
CTLA-4 deficient mice develop a massive and lethal lymphoproliferative disease that is more severe than similar phenotypes observed in Ipr mice, gld mice, mice with a T cell specific defect in TGFβ signal transduction or targeted deletion of the inhibitory molecule PD-1 (Chambers et al., Annu. Rev. Immunol. (2001), 19: 565-94). Absence of CTLA-4 results in an activated phenotype of peripheral T cells (Waterhouse et al., Science (1995) 10; 270 (5238): 985-8, Tivol et al., Immunity (1995) 3(5):541-7) whereas thymocyte development appears to be normal (Chambers et al., Proc. Natl. Acad. Sci. USA. (1997) 94(17):9296-301). From these observations it was concluded that CTLA-4 is necessary to regulate peripheral T cell tolerance and homeostasis of CD4+ and CD8+ T cells as polyclonal expansion of both populations occurs. The absence of CTLA-4 is most evident during the secondary responses in CTLA-4−/− TCR-transgenic models (Chambers C A et al Proc. Natl. Acad. Sci. USA. (1999) 96(15): 8603-8).
Several molecular mechanisms by which CTLA-4 inhibition occurs have been proposed including direct effects on phosphorylation levels, indirect effects due to competition with CD28 for ligand, sequestration of signalling molecules or disruption of signalling complexes (Chambers et al., Annu Rev Immunol. 2001; 19:565-94, Egen et al., Nat. Immunol. (2002) 3(7):611-8, Chikuma and Bluestone, Mol. Interv. 2002 2(4):205-8). Although the identity of the phosphatases involved are still debated, decreased phosphorylation of proximal TCR signalling molecules like CD3 ζ, EKR and JUN-N-terminal kinase have been observed when CTLA-4 cross-linking was used experimentally as CTLA-4 agonist. CTLA-4 might function at least in part by competing with CD28 for B7 ligands and thereby attenuating co-stimulatory signals indirectly particularly when B7 levels are low. Direct signalling through the tail of CTLA-4 appears to be necessary when B 7 levels are high which is further supported by the fact that a tailless CTLA-4 mutant on the cell surface of transgenic T cells in CTLA-4−/− mice delayed but did not prevent T cell activation and lymphoproliferation. The third model proposes that CTLA-4 physically disturbs the assembly or organization of molecules in the immunologic synapse. Formation of stable CTLA-4/B7 lattices due to the possible interaction of one CTLA-4 molecule with two B7 dimers as suggested by crystal structures may disturb the organized assembly of key components involved in the generation of TCR/CD28 signals.
CTLA-4 blockade with monoclonal antibodies or antibody fragments has been shown to lead to the rejection of a number of immunogenic transplantable tumor cell lines including colorectal carcinoma, renal carcinoma, lymphoma and fibrosarcoma cell lines (see for example, U.S. Pat. No. 6,682,736, US patent application 2002/0086014 or International patent application WO 01/14424). Less immunogenic tumor cell lines required concurrent combination therapy with a tumor vaccine, low dose of chemotherapy or surgical resection. The anti-tumor response elicited by CTLA-4 blockade is directed also towards normal tissue-derived proteins as autoimmune reactions were observed in mouse tumor models (B 16 melanoma, TRAMP tumor cell) and clinical trials. Recent phase I and II studies with human monoclonal antibodies are encouraging and the concurrent development of autoimmune reactions appears to be clinically manageable and might even correlate with therapeutic efficacy (Phan et al., Proc. Natl. Acad. Sci. USA (2003), 100: 8372-77, Sanderson et al. (2005), J. Clin. Oncol. 23: 741-50, Attia et al. (2005), J. Clin. Oncol. 23: 6043-53). On the other hand, recent results support the notion that enhanced tumor immunity through CTLA-4 blockade does not necessarily have to be linked with increased autoimmunity (Hodi et al., Proc. Natl. Acad. Sci USA (2003), 100: 4712-17, Lute et al., Blood (2005), 106(9): 3127-33). In addition to the application in cancer therapy, the use of CTLA-4 binding immunoglobulins for the treatment of infectious diseases and or auto-immune diseases is subject of intensive research.
However, antibodies and fragments thereof may not be suitable for all potential applications. One limiting factor may be their rather large molecular size, which is the case not only for intact antibodies but also for their antigen-binding fragments such as Fab fragments.
For this reason, alternatives to CTLA-4 blocking antibodies have been considered soon after the therapeutic potential of these antibodies emerged. International patent application WO 90/33770 is generally directed to ligands for T cell surface molecules, especially CTLA-4, which induces antigen specific apoptosis of activated T cells. Isolated peptides containing CTLA-4 fragments, constituting the epitope for such binding, are also disclosed and claimed. U.S. Pat. No. 6,337,316 discloses peptidometics capable of inhibiting CD28 and/or CTLA-4 interaction with CD80 (B7-1) and CD86 (B7-2) and having the core amino acid sequence Leu-Met-Tyr-Pro-Pro-Tyr-Tyr (SEQ ID NO: 36). An alternative to CTLA-4 blocking antibodies are recombinant lipocalins which bind CTLA-4 (see WO2006056464 and Schönfeld et al. (2009), Proc. Natl. Acad. Sci. USA 106(20): 8198-8203).
Despite these approaches, it would still be desirable to have further alternatives, yet even improved molecules that bind CTLA-4, for example for blocking the CTLA-4 interaction, and can be used in pharmaceutical and/or diagnostical applications as described above. It would also be desirable to have a compound that has an improved efficacy. Accordingly, it is an object of the present invention to provide such compounds.